3种鱼类弹状病毒荧光定量环介导等温扩增检测方法的建立  

Establishment of 3 fish rhabdovirus real-time fluorescent loop-mediated isothermal RNA amplification assay

作　　者：陈珍容[1] 陈进会[2] 陈洵 唐大运 石磊 颜其贵[1] 

机构地区：[1]四川农业大学动物医学学院,四川温江611130 [2]东莞出入境检验检疫局,广东东莞523072 [3]广州迪澳生物科技有限公司,广东广州510012

出　　处：《中国预防兽医学报》2015年第11期856-860,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：教育部<长江学者和创新团队发展计划>创新团队项目(IRTO848);2011年东莞市科技计划资助项目(2011108102047)

摘　　要：鲤春病毒血症病毒(SVCV)、病毒性出血性败血症病毒(VHSV)和传染性造血器官坏死病病毒(IHNV)是严重危害水产养殖业的3种鱼类弹状病毒。为建立这3种鱼类弹状病毒的快速检测方法,本研究设计合成针对这3种病毒系列引物,并在扩增反应体系中加入荧光染料SYBR Green I,通过恒温荧光检测仪Deaou-308C扩增检测,分别建立了这3种病毒的荧光定量环介导等温RNA扩增(RT-LAMP)方法。经反应条件的优化,检测结果显示,这3种方法分别对SVCV、VHSV和IHNV 3种弹状病毒具有特异性的扩增反应,对传染性胰坏死病毒、狗鱼弹状病毒和牙鲆弹状病毒核酸的扩增结果均为阴性,具有良好的特异性。灵敏度试验最低检测限分别为4.0×104拷贝/μL、5.4×104拷贝/μL、4.3×104拷贝/μL。该方法在63℃下保温40 min可以完成检测,操作简单、灵敏度高、特异性强,通过恒温实时荧光检测仪可实时检测及自动判读检测结果,适合现场检测和口岸快速检测。Spring viremia of carp virus （SVCV）, viral hemorrhagic septicemia virus （VHSV） and infectious hematopoietic necrosis virus （IHNV） are the mainly 3 kinds of fish rhabdovirus seriously affecting the aquaculture industry. To establish the three kinds of fish rhabdovirus detection assay, three real-time fluorescence loop-mediated isothermal RNA amplification （LAMP） detections based on SYBR Green I were developed with 3 sets of primers targeting the genes of SVCV, VHSV and IHNV, respectively. After optimizing the reaction conditions, the methods were specific for amplifications of the 3 rhabdovirus, respectively, but had no any amplification for pancreatic necrosis virus, pike rhabdovirus, flounder rhabdovirus. And the detection limit were 4.0×10^4 copies/uL for SVCV, 5.4×10^4 copies/uL for VHSV and 4.3×10^4 copies/uL for IHNV. The methods can be carried out at 63℃ for 40 rain. The results were able to be interpreted real-timely and automatically through a constant real-time fluorescent detector. The established assays are suitable for rapid detection of the 3 viruses.

关 键 词：鱼类弹状病毒 恒温荧光定量 环介导等温扩增 

分 类 号：S852.65[农业科学—基础兽医学]